The field is processes, systems, and instruments for automatically measuring transient activity in cells using image sequences. More specifically, the field includes use of image cytometry to automatically measure calcium transients in cardiomyocytes.
Automated image cytometry is increasingly used in high content screening (HCS) to automatically measure characteristics of objects in magnified images of cellular material. Typically, an automated image cytometry system includes tools to perform analysis of images and analysis of data acquired from the images. Image analysis may include staining material with one or more dyes, each selected to bind with a particular structure or component of the material. For example samples in a multi-well screening experiment related to obesity may be stained with one dye that binds specifically with cell nuclei, a second dye that binds specifically with cell cytoplasm, and a third dye that binds with lipid droplets. Magnified images of well contents may be selectively processed according to color in order to classify image objects such as nuclei, cell perimeters, and lipid droplets. Once classification is complete, data may be extracted from the classification results in order to quantify various properties of the objects. See, for example the related US publication 2008/0144895 A1.
Heart failure due to hypertension, infarction, or other factors, is a leading killer of men and women in modern society and involves, at its base, a debilitating loss of cardiomyocytes. Encouraging recent studies indicate the feasibility of regenerating lost cardiomyocytes, for example by transplanting embryonic stem cell-derived cardiac myocytes (ESCMs) or mobilizing resident stem cells. To realize the potential of stem-cell based therapies, it would be extremely beneficial to develop technology and instruments for high throughput, high content screening (HCS) of drugs and genes for their ability to stimulate the formation of functional, contractile cardiomyocytes.
The contractile cycle of cardiomyocytes shown in FIGS. 1 and 2 (adapted from Donald M. Bers, “Cardiac excitation-contraction coupling,” Nature, Vol 415, pp: 198-205, 2002) is divided into an abrupt shortening phase (systole), induced by a rapid rise in the intracellular calcium concentration ([Ca2+]i) due to calcium entry via voltage dependent L-type calcium channels and calcium-induced-calcium release from the sarcoplasmic reticulum (SR). Contraction is followed by relaxation of the cell (diastole) and the decline of [Ca2+]i. Decline of [Ca2+]i during diastole is controlled primarily by re-sequestration of calcium back into the SR by SERCA2, an ATPase associated with the SR membrane.
Calcium transients have been recorded from cardiomyocytes derived from embryonic stem cells (both from mouse and from human). For example, calcium transients in fura-2-loaded cardiomyocytes derived from human embryonic stem cells (hESC-CM), were observed in spontaneously contracting embryoid bodies [K. Dolnikov, et al., “Functional Properties of Human Embryonic Stem Cell-Derived Cardiomyocytes,” Ann. N.Y. Acad. Sci. 1047, pp: 66-75, 2005]. hESC-CMs cultured on mouse visceral-endoderm like cells showed similar calcium transients and spontaneous contractions at 0.6 to 1.5 Hz [Mummery et al., “Differentiation of Human Embryonic Stem Cells to Cardiomyocytes,” Circulation. 107, pp: 2733:2740, 2003]. Fluo-4 (a fluorescent dye) has been used to record calcium transients from murine ESCMs, which exhibited spontaneous contraction rates of approximately 1 Hz [Grey, et al., “Fine-tuning in Ca2+ homeostasis underlies progression of cardiomyopathy in myocytes derived from genetically modified embryonic stem cells,” Human Molecular Genetics 14(10), pp: 1367-1377, 2005]. Consistent with the wide spread observation of spontaneous beating in experiments designed to elicit the appearance of ESCMs, myocytes with “pace-maker” activity have been observed in contracting cell clusters, serving to drive the contractions of neighboring cells [Mery et al., “Initiation of Embryonic Cardiac Pacemaker Activity by Inositol 1,4,5-Trisphosphate-dependent Calcium Signaling,” Mol Biol Cell, 16(5), pp: 2414-23, 2005]. These studies indicate that ESCMs typically exhibit calcium transients and contractile characteristics similar to neonatal rat ventricular myocytes (NRVMs), rather than adult cardiomyocytes.
Automated high content screens allow for a wealth of information to be gathered from a given experimental study. If the hardware and controlling software are present, researchers may be able to fine tune hardware performance to directly match their experimental needs. Unfortunately, this is not always the case and researchers are usually forced to either tweak their experimental design or are required to build customized tools to conduct their experiments. Thus, it would be extremely desirable to be able to record Ca++ transients on a cell-by-cell basis in a manner that is easily integrated into current laboratory setups. A desirable Calcium Transient Image Cytometer (CTIC) would interface easily into available high content microscopy workstations which already perform multi-well plate scanning and image acquisition, to enable video burst acquisition and analysis of calcium transients in a fully automated (high throughput screening) mode. It is desirable that the CTIC electrically stimulate (or pace) the cells, record the resulting Ca++ transients from cells in microtiter plates (e.g., with 96 wells), and automatically quantify characteristics such as the duration of the Ca++ waves on a cell-by-cell basis in a fully automated manner on large scale screens (e.g., tens to hundreds of thousands of compounds).